In order to justify my reasons for administering certain drugs called lathyrogens topically into the site of fibrotic lesion, it is necessary to outline several topics related to this new therapeutical method.
Dynamics of Scar Formation
Scar callagen is synthesized by fibroblasts. While still within the fibroblasts, the collagen polypeptides are hydroxylated, form a triple helix and undergo glycosylation to the transport form, known as procollagen. Procollagen is then secreted from the fibroblasts into the extracellular wound environment, to form tropocollagen. Tropocollagen then undergoes intramolecular and intermolecular covalent cross-linking to form collagen fibers which are becoming progressively less soluble and acquiring mechanical strength. This process is also called maturation or polymerization of collagen and is the main target of this invention.
Formation of a scar is the result of a fibroproliferative inflammation. Scar is an imperfect method of repairing tissue defects. Several steps could be identified in the course of fibrotic reaction. The injury activates fibroblasts to produce more collagen and glycosaminoglycans. Often part of the injury is bleeding by ruptured vessels. Blood is a known factor inducing fibrosis. In ruptured or injured tendons, the presence of blood and blood proteins, mainly of fibrin, forms bridges between the tendon and its sheath to form peritendineous adhesions which immobilize the joint by impairing the gliding function of the tendon. Once collagen is formed and accumulates at the site of the injury, it matures and only then do the abnormal functions of the tissue manifest themselves.
In the dynamics of the fibroproductive inflammation, various reactions reach their maximum and then decline with time. Activity of lysyl oxidase, the enzyme which is involved in polymerization of collagen, is highest at much later stages after collagen has been accumulated in the intercellular space. The maximal activity of lysyl oxidase coincides with formation of an insoluble collagen within the injured tissue. Consequently, the optimal time of "pharmacological" interference with a certain step of the inflammation is at the time of maximum incidence and activity of this specific process. We learned by our experiments that in skin incision wounds, the inhibition of lysyl oxidase could be started five days after inflicting the injury, and continued the treatment for 14-21 days to achieve an effective and permanent prevention of collagen maturation.
The fibrotic collagenous tissue is in a continuous increased metabolic turnover. Both synthesis and degradation of collagen are increased and their relative activities change with time of healing. It could be assumed that by decreasing the structural stability in the scar (i.e., decreasing the degree of polymerization of collagen) by administration of lathyrogenic agent, the pool of "extractable" collagen is increased. These forms would be more readily available to digestion by collagenase system and by other proteolytic enzymes in the later stage. This will result in a reduction of the size and volume of the fibrotic mass.
Pharmacology of Fibrosis
There exist several steps unique for collagen which could be used as a target for so-called "specific interference" with collagen metabolism. These stages are:
1. Hydroxylation of prolyl and lysyl residues by appropriate hydroxylases. PA1 2. Glycosylation of some .epsilon.-NH.sub.2 hydroxylysyl groups and; PA1 3. subsequent secretion of the molecule out of the cell. PA1 4. In the extracellular space, the molecule further polymerizes or matures under the effect of lysyl oxidase which forms the basis for development of stable covalent cross-links. PA1 5. At different stages of collagen synthesis the molecule is degradable but the "younger" the molecule is, the faster the degradation by tissue collagenases. PA1 (a) polar-nonpolar nature of the substance PA1 (b) hydration of the skin PA1 (c) blood supply PA1 (d) modification of the stratum corneum by chemicals.
Several sophisticated methods were developed and studied with the aim to reduce collagen deposition or polymerization in the fibrotic lesion (D. J. Prockop German Pat. No. 2,228,187). In all these situations, the medication was administered either perorally or parenterally, in other words by systemic route, where the drug was distributed among all tissue and body fluids.
All various methods of interference with individual steps of collagen synthesis have one major deficiency; they work very nicely in isolated, closed systems of cells in tissue cultures and are minimally effective or quite ineffective in vivo in the whole organism. In fact, after systemic administration of many of these drugs (proline analogs, chelating agents, colchicine, etc.) their toxic effect is close to the therapeutic effect. Thus, there is a permanent risk of general toxicity of the drug used.
The only exception seems to be the use of a lathyrogen to interfere with collagen polymerization mainly because of high effectiveness of a typical representative of lathyrogens, beta-aminopropionitrile (BAPN), to inhibit lysyl oxidase at 10.sup.-7 M concentrations. Still, the fast metabolism of BAPN requires frequent administration of this potent drug (every 6 hours seems to be optimal) which may result in induction of some toxic adverse effects in both lab animals as well as in humans.
Maturation (Polymerization of Collagen)
The enzyme forming the basis for the polymerization of collagen is lysyl oxidase. Lysyl oxidase oxidatively deaminates specific .epsilon.-amino groups of peptidyl lysine and hydroxylysine residues contained within the structural proteins collagen and elastin. The aldehyde product then forms, nonenzymatically, either Schiff base adducts with other specific .epsilon.-amino peptidyl lysine or hydroxylysine residues or forms aldol condensates with other preformed aldehydric components. These cross-linking reactions lend structural integrity to collagenous and elastinous connective tissue.
In principle, we may interfere directly with the function of lysyl oxidase by various lathyrogenic agents, BAPN and aminoacetonitrile being the most effective. Inhibition of lysyl oxidase by BAPN is irreversible. As will be shown below, lysyl oxidase has very high turnover and its activity in a granuloma tissue after a single systemic moderate dose of BAPN recovers within 6-12 hours. Another possibility is blocking the formed aldehydes, as with D-penicillamine, thus preventing condensation of aldehydes or formation of Schiff bases as shown in Scheme I. ##STR1## Since BAPN and D-penicillamine affect two different sites in the formation of covalent cross-links, when used together their effect is additive. The lathyritic properties of penicillamine derive from its ability to chelate aldehydes formed by the action of lysyl oxidase on the epsilon amino group of tropocollagen lysine. In addition, direct inhibition of of lysyl oxidase by D-penicillamine was demonstrated. The overall effectiveness of systemically-administered D-penicillamine in decreasing the structural stability of collagen has been well-documented. All of the clinical studies using the treatment with D-penicillamine have indicated a high incidence of acute hypersensitivity reactions. Striking changes in the metal content in various tissues and fluids after D-penicillamine treatment were reported. It is mainly the metabolism of zinc and copper which is affected and which results in various deficiencies and pathologies. These symptoms are suppressed by systemic steroids; thus, a combination of D-penicillamine and prednisone should minimize toxic side effects. Topical administration of D-penicillamine by injection route or by percutaneous absorption similar to BAPN uses several times lower dose than that used systemically. This avoids the incidence of toxic complications.
Toxicity of Systemically-Administered Lathyrogens
The systemic toxicity of BAPN has been the major obstacle in using this drug in large scale in human pathology. There is no doubt that at lathyrogenic dosages of BAPN, the animals stop growing, lose body weight and lower their food intake. Also, the animal's behavior is changed--they do not clean their fur and are irritated if they are touched. All this indicates a general toxicity. There is also no doubt that various lathyrogenic drugs, mainly BAPN showed to be very effective in the blocking the polymerization of collagen in various animal models of fibrotic lesion.
Because of success in animal studies, Keiser and Sjoerdsma (1967) studied the effect of BAPN in patients with fibrotic lesions of the skin and other organs called scleroderma. In short-term treatment with a dose of 2 g/day, no toxic side effects were noted; longer courses of treatment were, however, associated with prohibitive reactions, such as allergic skin rash and hemolytic anemia. Peacock and Madden (1969) employed BAPN in clinical trials with humans undergoing flexor tendon surgery. Although a significant number of the patients exhibited hypersensitivity to the agent, decreased covalent bonding of human collagen with significant clinical benefit was demonstrated.
One way to reduce the systemic toxicity of BAPN without interfering with its lathyrogenic activity is to inhibit the metabolism of this drug. If degradation of BAPN is blocked by pargyline, a monoamine oxidase (MAO) inhibitor, prolonged lysyl oxidase inhibition potentiates wound strength diminution without accompanying toxicity. Indeed, isoniazid (INH), both a weak lysyl oxidase and MAO inhibitor potentiate the lathyritic effects of BAPN. The addition of isoniazid or pargyline to an otherwise effective dose of BAPN profoundly inhibited lysyl oxidase and depressed wound burst strength. All animals gained weight throughout the experiment.
In order to overcome the general toxicity of systemically administered BAPN, I studied two methods, both of which showed to be unsuccessful. In the first method BAPN was bound by amide- and ionic linkage to a polymer such as polyacrylic acid, with the aim to obtain long lasting sustained release of the drug. It was found, however, that many carriers for BAPN induced nonacceptable fibrotic tissue reaction when injected in injured tissues. The second approach intended to increase the percutaneous absorption of BAPN by synthesizing BAPN base. Indeed it was demonstrated that BAPN base penetrates the skin barrier approximately five times faster than BAPN fumarate. Unfortunately, the base showed to be unstable under storage, undergoing hydrolysis forming ammonia and toxic acrylonitrile. For these reasons we discontinued the use of either polymer-BAPN fumarate complex of BAPN-base.
Based on the evidence of systemic toxicity of BAPN, I arrived at the conclusion that the only method of preventing the toxic problems of this clinically important and needed drug is to administer BAPN and other lathyrogens topically. There exist several clinical symptoms with collagen pathology, which could be treated with benefit by topically administered BAPN. These include skin scar contractures, such as after deep burns, peritendineous adhesions after injury to the tendon, perineural adhesions, stiffness of periarticular tissue of immobilized joints and Dupuytren's contracture and other fibrotic lesions common in human and veterinary medicine.
Although the pathogenesis of the above disorders may differ, in every one it was postulated that the physical properties of the scar tissue were affected by abnormal cross-linking of the collagenous component. A variety of methods was proposed and used to alleviate the problems of abnormal reactivity of polymerized scar tissue (Peacock and Van Winkle 1976). In all the above manifestations BAPN systemic administration was suggested. The ideal method of topical administration would be the noninvasive percutaneous resorption of BAPN painted onto the skin as proposed in this disclosure. I will show that BAPN is transported through the skin and effectively inhibits lysyl oxidase in granuloma tissue formed in subcutaneously implanted polyvinyl alcohol sponge. At the same time, no morphological or ultrastructural evidence of topical cytotoxicity is found. These findings assured me that topical BAPN is safe, effective and optimal therapeutic regimen to achieve significant interference with maturation of collagenous structure in tissues with an injury.
Peritendineous adhesions as well as stiffness is injured or immobilized joints is a major clinical problem. Complete rehabilitation of a patient may be prolonged or prevented by such adhesions, scar contractures or joint stiffness. The lesions are due to the formation and remodeling of collagen within scar, ligaments, or fascia. It was shown experimentally in rats and dogs that inhibition of collagen cross-linking in and around tenolyzed tissue or an immobilized joint using systemically-administered BAPN could significantly improve the tendon gliding fraction or reduce stiffness. In both species, however, the systemic administration of the lathyrogen resulted in undesirable side effects. Still, the beneficial effects on reducing adhesions or joint stiffness have encouraged my efforts to introduce the lathyrogen locally at the site of the tendon lesion or over the immobilized joint by topical administration.
Percutaneous Resorption
This disclosure proposes to inhibit collagen in specific fibrosis related lesions either by topical application of BAPN or other lathyrogens onto the skin or by direct injection into the lesion. The major limitation of topical drug administration onto the skin surface is the imposing impermeability of the skin, which forms a chemical and waterproofing seal. By several studies it was shown that the rate of percutaneous resorption is limited mainly by the stratum corneum, formed by 15 cells thick layer of cornified cells. Substantial knowledge has been accumulated and reviewed on the permeability of the skin.
It was shown that the rate of transport of some solutes through the skin depends on:
Accordingly, non-charged molecules penetrate faster. I demonstrated this by showing higher penetration of BAPN-base than that of BAPN-fumarate. The instability of the base is, however, the major obstacle in using this drug. Occlusive bandage with the solutes enhances penetration considerably. UV-irradiation of the skin, causing erythema, is supporting percutaneous resorption. Detergents, dimethylsulfoxide (DMSO) and several other solvents have been shown to substantially increase the process. Beta-aminopropionitrile (BAPN), as the term is used herein, includes both free BAPN and BAPN salts, such as BAPN-fumerate, which also function to inhibit lysyl oxidase. The various solvents and solvent mixtures, including aqueous buffers, and water-containing solvent systems, such as buffer/DMSO, which are used to carry BAPN for topical administration are also referred to herein as drug carriers. It will be understood by those skilled in the art that such carries may be formulated to contain emulsifiers or the like for use particularly in compounding the BAPN for external topical, i.e., percutaneous administration. The drug and its drug carrier are referred to, collectively, as a drug preparation.
This disclosure will demonstrate that BAPN-fumarate effectively penetrates the stratum corneum barrier, and is excreted into urine, if administered onto the pretreated skin by a method described by this invention. The most important is, however, that due to this transport, BAPN inhibits collagen maturation (lysyl oxidase activity) in the subcutaneously induced granuloma tissue. This makes it possible to use the simplest method of BAPN administration, i.e., painting the pretreated skin with BAPN base fluid in combination with occlusive dressing or to use special delivery system as consisting of hydrophilic pouch containing the solution of the drug described in this disclosure.